Any discussion of the background art throughout the specification should in no way be considered as an admission that such background art is prior art, nor that such background art is widely known or forms part of the common general knowledge in the field.
Fluorescence is the name given to the process of light emission following a transition between energy levels that occur without a change in the electron spin state. It is typically a short-lived phenomenon with excited-state lifetimes measured in nanoseconds for most common fluorophores. Phosphorescence on the other hand describes excited state transitions that involve a change in spin-state, and when these transitions are spin-forbidden, emission lifetimes can extend for several thousand times longer than fluorescence decay.
Fluorescence based techniques provide a powerful means for both the qualitative and quantitative detection of bio-molecules. Fluorescence methods afford a sensitive means for the detection of single molecules, however, fluorescent probes (also referred to as fluorescent markers), used to “label” a particular feature in a sample (for example a particular organism or bio-molecule such as Giardia in a water sample) to determine the presence and/or number of features in the sample, lose much of their discriminatory power in the presence of autofluorescence. Organic and inorganic autofluorophores are ubiquitous in nature and some materials fluoresce with great intensity, obscuring or diminishing the visibility of synthetic fluorescent probes. Spectral selection techniques (emission and excitation filters) can reduce the problem but are not always applicable due to the abundance and spectral range of autofluorescence.
Using techniques such as time-resolved fluorescence microscopy (TRFM), it is possible to spatially discriminate fluorescent regions that differ by less than a nanosecond in fluorescence lifetime. TRFM techniques operate in the frequency domain, usually employing a sinusoidal modulation of the excitation source to induce a phase (φ) delayed modulation in fluorescence intensity from which fluorescence lifetime can be determined using Δφ and modulation frequency parameters (FIG. 1A).
Time-gated luminescence (TGL) techniques operate within the time-domain and are directed towards detection of events that occur at much longer time-scales (phosphorescence).
For TGL operation as shown in FIG. 1B, the detector is gated off whilst a brief pulse of light is used to excite emission from the sample. The detector is maintained in the off-state for a resolving period (gate-delay) whilst short-lived (<1 μs) fluorescence fades beyond detection. The detector is then enabled to capture luminescence in the absence of autofluorescence, greatly increasing the signal-to-noise ratio. As can be seen, pulse fluorometry or time-gated luminescence techniques operate on much longer timescales. Fluorescence lifetime constants can be determined by observing intensity in the fluorescence signal after different gate delays. A key advantage of TGL techniques is the suppression of short-lived fluorescence to increase the signal-to-noise ratio (SNR).
Whilst it is possible to discriminate probe fluorescence from autofluorescence using TRF techniques, a simpler and much less costly TGL instrument can be employed if a suitable luminescent probe is available. Lanthanide (Eu3+ or Tb3+) chelate luminescent probes have exceptionally long phosphorescence lifetimes (τ) reaching milliseconds for some compounds. Other compounds that have found wide application for TGL include the platinum and palladium (copro)porphyrins with lifetimes ranging from 10 to 1000 μs depending on their environment. The very large difference in τ between typical autofluorophores and the luminophores used for TGL has helped ensure useful results were gained even with simple instruments relying upon chopper-wheels.
The substantial increase in SNR afforded by TGL techniques is a critical factor when searching for rare target organisms encountered in autofluorescent environments. For example, the detection of Cryptosporidium oocysts in drinking water requires the filtration of large volumes of water and results in a matrix of mineral particles, algae, desmids and plant matter that is strongly autofluorescent. TGL microscopy has been demonstrated to greatly suppress this background and simplify the detection of both Giardia and Cryptosporidium, two important waterborne pathogens. There are instances where the detection of rare-event signals using conventional fluorescence techniques is exceedingly difficult (or impossible) and consequently, where TGL microscopy has greatest utility.
Luminescent probes based on the lanthanides Eu3+ and Tb3+ were described in the 1960's but effective immunofluorophores using these compounds were not reported until the early 1980's. TGL microscopes were built to exploit these novel compounds however various deficiencies in the instrumentation and luminescent probes resulted in relatively insensitive instruments. As technologies matured, improvements were made both in instrument design and probe quality. The evolution of TGL microscope instrumentation designed for the detection of phosphorescence (τ>10 μs) is briefly discussed below.
With reference to FIG. 1B, TGL techniques employ an excitation pulse to excite photon emission from the sample. The excitation pulse ideally terminates abruptly (<1 μs) whilst the detector is maintained in the off-state for a predetermined time (the resolving period or gate delay), the duration of which is designed to permit short-lived fluorescence to decay. After the gate-delay has elapsed, the detector is gated on to initiate the start of the acquisition period to detect the fluorescence signal from the luminescent label, free of background noise from autofluorophores in the sample.
In 1988 Soini et al. [Soini E J, Pelliniemi L J, Hemmila I A, Mukkala V M, Kankare J J, Frojdman K, Lanthanide chelates as new fluorochrome labels for cytochemistry, Journal of Histochemistry and Cytochemistry 36(11) 1988 p. 1449-51] described a europium chelate that could be easily bound to bio-molecules to permit them to “re-test the old idea of time-resolved fluorescence microscopy in immunohistology and cytology”. Using steady state excitation, it was shown that Eu-antibody labelled histology sections were visibly luminescent to the naked eye and would likely provide a means to improve signal to noise ratio under TGL conditions. The following year, Beverloo et al. described a Xenon flashlamp excited TGL microscope synchronized to a chopper wheel. Phosphorescence persists for orders of magnitude longer than prompt fluorescence and makes feasible the use of mechanical choppers for visualizing the phenomenon and the majority of early TGL instruments employed chopper wheels to isolate the excitation and detection states in a TGL cycle.
The first TGL microscope employing two phase locked chopper-wheels for pulse control (detection and excitation) was reported by Marriott et al. in 1994. FIGS. 2A and 2B details the essential features of such an instrument 100. Excitation chopper wheel 101 and emission chopper wheel 103 are shown with the chopper-blades positioned in their respective states for the excitation and detection (emission) states of a TGL cycle FIGS. 2A and 2B respectively). During the excitation state of FIG. 2A light 105 from excitation light source 107 passes between the blades of the excitation chopper 103 and is deflected by a dichroic mirror 109 in filter cube 111 and is focused by microscope objective 113 onto a sample under test (not shown). During this state, the prompt or autofluorescence 115 from short-lived autofluorophores in the sample is directed back through the microscope objective 113, and through the dichroic mirror 109, but is blocked from reaching the detector (not shown) by the emission chopper 103 i.e. in this state the excitation light source is open and the detector is closed. FIG. 2B shows the detection (emission) state of the TGL chopper where the blades of choppers 101 and 103 have moved such that light 105 from the excitation source 107 is blocked by the blades of the excitation chopper 101 whereas the phosphorescence 117 from the luminescently labelled sample passes through the blades of emission chopper 103 to the detector.
The detection-side chopper 103 is phase locked to the excitation chopper 101 by a control module (not shown) to maintain an arbitrary phase difference between the two. The TGL system 100 can be switched from delayed luminescence mode to ‘prompt’ mode by adjusting this phase angle.
As described above, the gate-delay is the intervening period between termination of the excitation pulse and commencement of the acquisition phase (see FIG. 1B). For most phosphorescent labels, decay follows single exponential kinetics (I0=Ite−t/τ) and emission decays substantially as the gate-delay interval approaches the luminescence lifetime. Unfortunately, the gate-delays typical for chopper based switching mechanisms are quite long (about 20 to 500 μs) in comparison to the emission decay time of the fluorescent labels and therefore the fluorescence has typically decayed quite significantly before the detection state is able to commence. Also, conventional time-gated luminescence microscopes incur substantial losses both during the excitation and emission/detection states as light passages through the dichroic filter cube. The dichroic mirror in the filter cube also has significant limitations on the particular wavelengths that are allowed to be either reflected on to the sample or to pass through to be detected due to the limitations in coating designs. Thus, such systems have severe limitations on the number of allowed wavelengths and the reflection/transmission bandwidth of the design wavelengths that are able to be utilised for the detection system. Such dichroic filter cubes are also expensive and complex to interchange causing significant barriers to the use of the TGL microscope system for different excitation sources or luminescent probes with different operating wavelengths.
Electronic shutters may be used to overcome the gate-delay limitation of chopper-wheel systems, for example ferro-electric liquid crystals (FELC) which rotate the plane of light polarization in response to an applied voltage and can serve as fast optical shutters. A TGL microscope was constructed by Verwoerd et al. in 1994 [Verwoerd N P, Hennink E J, Bonnet J, Van der Geest C R, Tanke H J, Use of ferro-electric liquid crystal shutters for time-resolved fluorescence microscopy, Cytometry 16(2) 1994 p. 113-7] in which the emission-plane chopper was replaced with two crossed LC shutters. For excitation, a Xenon-arc lamp was interrupted by a mechanical chopper to generate pulsed output; gating of the LC shutters was synchronized to the chopper wheel position. Whilst effective, the FELC shutters imposed a substantial insertion loss with transmission reduced to just 15% when fully open. A further limitation of chopper excitation schemes arises from the relatively slow rise and fall time of the pulse (for example, in the systems described by Verwoerd et al, the rise/fall time was 50 to 100 μs at a chopper rotation speed of 3,800 rpm). Excitation pulses with slow falling edges force extension of the gate-delay that leads ultimately, to a loss in SNR. The gain achieved by switching rapidly in the emission plane with the FELC shutter was offset by the slow falling edge of the excitation pulse.
Since 1994, the majority of the improvements in TGL microscope detection systems have been obtained by improvements in either the excitation source—flashlamp, visible and ultraviolet (UV) lasers, or light emitting diodes (LEDs)—and/or the detectors used to capture the fluorescent light with increasingly improved signal-to-noise ratio—image-intensified gated charge coupled device (CCD) and electron multiplying charge coupled device (EMCCD) detectors have been particularly successful as described in the inventor's earlier patent application PCT/AU2005/001606.
The basic requirements for TGL microscopy are relatively straightforward, a pulsed excitation source and a gated detector. However, the common features of the prior art TGL microscope systems to provide these basic requirements has been the dual shutter system (either using chopper wheels, electronic shutters, or a combination of both) similar to that depicted in FIGS. 2A and 2B. As previously described, this type of system requires that the phase of each of the excitation- and emission-side shutters are precisely synchronised with both the excitation source and the detector acquisition state. To achieve this, complex electronic phase matching circuitry and control systems are required, which are both expensive to implement and maintain. Furthermore, the detector systems that must be used with these TGL microscope systems are prohibitively expensive (typical cost of a TGL EMCCD detector at the time of writing is in the range of about $20,000 to $45,000).
The prohibitive cost of such systems means that they are not available on a large scale since only the most well-funded research facilities can purchase and maintain such items.
Therefore, there is a need for a TGL system that is simple to both implement and use.